During development, homeostasis, tumor growth, or ischemia, cellular oxygen levels are often insufficient to meet physiological demands. The human hypoxia-inducible factor (HIF-1) implements most of the critical transcriptional changes that mediate adaptation to hypoxia. Pharmaceuticals that modulate HIF activity hold great therapeutic potential for preventing cancer growth and for mitigating the effects of cardiovascular disease. The long-term goals of this project are to identify and characterize the molecules and pathways that control HIF activity. The central hypothesis guiding these studies is that cells employ multiple strategies to monitor oxygen levels and keep HIF-1 activity "in check". Our experimental strategy employs Caenorhabditis elegans, a powerful system for gene discovery and characterization of genetic networks. This innovative approach will complement and extend the wealth of molecular and biochemical studies that have investigated the regulation of HIF in cancer cell lines. The C. elegans hif-1, egl-9 and vhl-1 genes are orthologous to the genes encoding the Hlp alpha subunit, the EGLN/ PHD / HPH prolyl hydroxylase, and the VHL tumor suppressor, respectively. In preliminary studies, we have shown that EGL-9 inhibits HIF-1 function via two distinct mechanisms: (i) the well-described EGL-9 / VHL-1 pathway for oxygen-dependent degradation of HIF-1, and (ii) a VHL-1-independent pathway for inhibition of HIF-1 transcriptional activity that remains to be characterized. Using forward genetic strategies, we have identified mutations in other genes (termed "rhy", regulators of hypoxia-inducible factor) that appear to act in concert with EGL-9 to regulate HIF-1 activity. The objective of this proposal is to identify and characterize genes and cellular networks that inhibit HIF transcriptional activity, independent of the EGL-9 /VHL-1 pathway for regulating HIF protein levels. Towards this goal, we propose three specific aims: (1) Characterize the molecular mechanisms by which EGL-9 inhibits HIF-1 activity, independent of VHL-1;(2) Identify and characterize genes that act in concert with EGL-9 or in parallel pathways to attenuate HIF-1 activity;(3) Define RHY-1 topology and elucidate the role of RHY-1 in HIF-1 regulatory networks. These studies will provide important insights to HIF regulatory networks and will advance the development of therapies to treat cancer and heart disease.